METHODS FOR REDUCING LIPOPROTEIN(a) LEVELS BY ADMINISTERING AN INHIBITOR OF PROPROTEIN CONVERTASE SUBTILISIN KEXIN-9 (PCSK9)

ABSTRACT

The present invention provides methods for reducing lipoprotein(a) (Lp(a)) in patients. The methods of the present invention comprise selecting a patient who exhibits elevated serum Lp(a), and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody such as the exemplary antibody referred to herein as mAb316P.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application Nos. 61/535,392, filed on Sep. 16, 2011;61/559,162, filed on Nov. 14, 2011; and 61/641,321, filed on May 2,2012, the disclosures of which are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic treatments ofdiseases and disorders which are associated with elevated levels oflipoproteins. More specifically, the invention relates to theadministration of PCSK9 inhibitors to reduce the levels of serum Lp(a)in a patient.

BACKGROUND

Lipoprotein(a) (Lp(a)) is a low-density lipoprotein-like particle formedby the association of apolipoprotein(a) (Apo(a)) with apoplipoproteinB100 (ApoB100). The Apo(a) component is covalently linked to the ApoB100component in the assembled Lp(a) particle via a disulfide bond. Elevatedserum Lp(a) has been shown in several studies to correlate with avariety of atherosclerotic and thrombotic disorders. (See, e.g.,Marcovina and Koschinsky (1998), Am. J. Cardiol. 82:57 U-66U; Ignatescuet al. (1998), Thromb. Haemost. 80:231-232; Lippi and Guidi (2000), Q.J. Med. 93:75-84; Bennet et al. (2008), Arch. Intern. Med. 168:598-608;The Emerging Risk Factors Collaboration (2009), J. Am. Med. Assoc.302:412-423; and Lamon-Fava et al, (2011), J. Lipid Res. 52:1181-1187).Thus, therapeutic reduction of serum Lp(a) levels has been suggested asa means for treating or reducing the risk of cardiovascular disorders.There are few available therapeutic options for lowering serum Lp(a)levels. Examples of treatments that have been tested and/or proposed forlowering serum Lp(a) include administration of acetylsalicylic acid,L-carnitine, niacin or anacetrapib, or LDL apheresis. (See, e.g.,Parhofer (2011), Curr. Pharm Des 17:871-876) No currently availabletreatments, however, provide adequate and practical therapy for elevatedLp(a).

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a proproteinconvertase belonging to the proteinase K subfamily of the secretorysubtilase family. The use of PCSK9 inhibitors (anti-PCSK9 antibodies) toreduce serum total cholesterol, LDL cholesterol and serum triglyceridesis described in US Patent Appl. Publ. No. 2010/0166768. Nonetheless,heretofore, PCSK9 inhibitors have not been shown to lower Lp(a) levelsin patients. Thus, there remains a need in the art for therapeuticmethods of lowering serum Lp(a) levels.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses the aforementioned need in the art byproviding methods for lowering serum Lp(a) levels in an individual. Themethods of the invention comprise selecting a patient who exhibitselevated serum Lp(a), and administering a pharmaceutical compositioncomprising a PCSK9 inhibitor to the patient. The patient is selected onthe basis of having an elevated serum Lp(a) level that is indicative ofenhanced risk for cardiovascular and/or thrombotic occlusive diseasesand disorders. The patient may also be selected on the basis ofexhibiting additional risk factors for such diseases and disorders inwhich a reduction in Lp(a) levels would be beneficial or risk-lowering.For example, patients with hypercholesterolemia (e.g. heFH, nonFH, etc.)may be good candidates for treatment with the therapeutic methods of thepresent invention.

PCSK9 inhibitors which may be administered in accordance with themethods of the present invention include, e.g., anti-PCSK9 antibodies orantigen-binding fragments thereof. Specific exemplary anti-PCSK9antibodies which may be used in the practice of the methods of thepresent invention include any antibodies or antigen-binding fragments asset forth in US Patent Appl. Publ. No. 2010/0166768, and/or disclosedherein.

The PCSK9 inhibitor may be administered to a subject subcutaneously orintravenously. Furthermore, the PCSK9 inhibitor may be administered to apatient who is on a therapeutic statin regimen at the time oftherapeutic intervention.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to describe intheir entirety.

Elevated Serum Lp(a) Levels

The present invention provides methods for reducing serum Lp(a) levelsin a patient. The methods of the invention comprise selecting a patientwho exhibits elevated serum Lp(a), and administering to the patient apharmaceutical composition comprising a PCSK9 inhibitor. As used herein,the expressions “Lp(a)” or “lipoprotein(a)” refer to a low-densitylipoprotein-like particle formed by the association of apolipoprotein(a)(apo(a)) with apolipoprotein B100 (apo B100).

In the context of the present invention, “elevated serum Lp(a)” means aserum Lp(a) level greater than about 14 mg/dL. In certain embodiments, apatient is considered to exhibit elevated serum Lp(a) if the level ofserum Lp(a) measured in the patient is greater than about 15 mg/dL, 20mg/dL, 25 mg/dL, 30 mg/dL, 35 mg/dL, 40 mg/dL, 45 mg/dL, 50 mg/dL, 60mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL, 100 mg/dL, 120 mg/dL, 140 mg/dL,160 mg/dL, 180 mg/dL, or 200 mg/dL. The serum Lp(a) level can bemeasured in a patient post-prandial. In some embodiments, the Lp(a)level is measured after a period of time of fasting (e.g., after fastingfor 6 hrs, 8 hrs, 10 hrs, 12 hrs or more). An exemplary method formeasuring serum Lp(a) in a patient is by rate immune-nephelometry,although any clinically acceptable diagnostic method can be used in thecontext of the present invention.

Patient Population

The methods of the present invention are useful for reducing serum Lp(a)levels in human subjects that exhibit an elevated level of serum Lp(a).In some instances the patient is otherwise healthy except for exhibitingelevated serum Lp(a). For example, the patient may not exhibit any otherrisk factor of cardiovascular, thrombotic or other diseases or disordersat the time of treatment. In other instances, however, the patient isselected on the basis of being diagnosed with, or at risk of developing,a disease or disorder that is caused by or correlated with elevatedserum Lp(a). For example, at the time of, or prior to administration ofthe pharmaceutical composition of the present invention, the patient maybe diagnosed with or identified as being at risk of developing acardiovascular disease or disorder, such as, e.g., coronary arterydisease, acute myocardial infarction, asymptomatic carotidatherosclerosis, stroke, peripheral artery occlusive disease, etc. Thecardiovascular disease or disorder, in some instances, ishypercholesterolemia. For example, a patient may be selected fortreatment with the methods of the present invention if the patient isdiagnosed with or identified as being at risk of developing ahypercholesterolemia condition such as, e.g., heterozygous FamilialHypercholesterolemia (heFH), homozygous Familial Hypercholesterolemia(hoFH), as well as incidences of hypercholesterolemia that are distinctfrom Familial Hypercholesterolemia (nonFH).

In other instances, at the time of, or prior to administration of thepharmaceutical composition of the present invention, the patient may bediagnosed with or identified as being at risk of developing a thromboticocclusive disease or disorder, such as, e.g., pulmonary embolism,central retinal vein occlusion, etc. In certain embodiments, the patientis selected on the basis of being diagnosed with or at risk ofdeveloping a combination of two or more of the above-mentioned diseasesor disorders. For example, at the time of, or prior to administration ofthe pharmaceutical composition of the present invention, the patient maybe diagnosed with or identified as being at risk of developing coronaryartery disease and pulmonary embolism. Other diagnostic combinations(e.g., atherosclerosis and central retinal vein occlusion, heFH andstroke, etc.) are also included in the definition of the patientpopulations that are treatable by the methods of the present invention.

In yet other instances, the patient who is to be treated with themethods of the present invention is selected on the basis of one or morefactors selected from the group consisting of age (e.g., older than 40,45, 50, 55, 60, 65, 70, 75, or 80 years), race, gender (male or female),exercise habits (e.g., regular exerciser, non-exerciser), otherpreexisting medical conditions (e.g., type-ii diabetes, high bloodpressure, etc.), and current medication status (e.g., currently takingstatins [e.g., cerivastatin, atorvastatin, simvastatin, pitavastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.], betablockers, niacin, etc.). The present invention also includes methods forreducing serum Lp(a) levels in patients who are intolerant of,non-responsive to, or inadequately responsive to conventional statintherapy. Potential patients can be selected/screened on the basis of oneor more of these factors (e.g., by questionnaire, diagnostic evaluation,etc.) before being treated with the methods of the present invention.

The present invention also includes methods for increasingtransintestinal cholesterol excretion (TICE) in a subject byadministering a PCSK9 inhibitor to the subject. For example, the presentinvention provides methods for increasing TICE in a subject byadministering to the subject an anti-PCSK9 antibody, e.g., the antibodyreferred to herein as “mAb316P”. According to certain embodiments, thepresent invention includes methods comprising identifying a subject forwhich enhanced TICE would be beneficial, or identifying a subject thatexhibits impaired TICE, and administering a PCSK9 inhibitor (e.g.,mAb316P) to the subject.

PCSK9 Inhibitors

The methods of the present invention comprise administering to a patienta therapeutic composition comprising a PCSK9 inhibitor. As used herein,a “PCSK9 inhibitor” is any agent which binds to or interacts with humanPCSK9 and inhibits the normal biological function of PCSK9 in vitro orin vivo. Non-limiting examples of categories of PCSK9 inhibitors includesmall molecule PCSK9 antagonists, peptide-based PCSK9 antagonists (e.g.,“peptibody” molecules), and antibodies or antigen-binding fragments ofantibodies that specifically bind human PCSK9.

The term “human proprotein convertase subtilisin/kexin type 9” or “humanPCSK9” or “hPCSK9”, as used herein, refers to PCSK9 having the nucleicacid sequence shown in SEQ ID NO:754 and the amino acid sequence of SEQID NO:755, or a biologically active fragment thereof.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-PCSK9 antibody (or antigen-binding portion thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(N) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The constant region of an antibody is important in the ability of anantibody to fix complement and mediate cell-dependent cytotoxicity.Thus, the isotype of an antibody may be selected on the basis of whetherit is desirable for the antibody to mediate cytotoxicity.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay nonetheless include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo),for example in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody” for purposes of the presentinvention. An isolated antibody also includes an antibody in situ withina recombinant cell. Isolated antibodies are antibodies that have beensubjected to at least one purification or isolation step. According tocertain embodiments, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Methods for determiningwhether an antibody specifically binds to an antigen are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. For example, an antibody that “specificallybinds” PCSK9, as used in the context of the present invention, includesantibodies that bind PCSK9 or portion thereof with a K_(D) of less thanabout 1000 nM, less than about 500 nM, less than about 300 nM, less thanabout 200 nM, less than about 100 nM, less than about 90 nM, less thanabout 80 nM, less than about 70 nM, less than about 60 nM, less thanabout 50 nM, less than about 40 nM, less than about 30 nM, less thanabout 20 nM, less than about 10 nM, less than about 5 nM, less thanabout 4 nM, less than about 3 nM, less than about 2 nM, less than about1 nM or less than about 0.5 nM, as measured in a surface plasmonresonance assay. An isolated antibody that specifically binds humanPCSK9, however, have cross-reactivity to other antigens, such as PCSK9molecules from other (non-human) species.

The anti-PCSK9 antibodies useful for the methods of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes methodsinvolving the use of antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. The use of antibodies and antigen-bindingfragments obtained in this general manner are encompassed within thepresent invention.

The present invention also includes methods involving the use ofanti-PCSK9 antibodies comprising variants of any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein having one or moreconservative substitutions. For example, the present invention includesthe use of anti-PCSK9 antibodies having HCVR, LCVR, and/or CDR aminoacid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 orfewer, etc. conservative amino acid substitutions relative to any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind tohuman PCSK9.

Using VELOCIMMUNE™ technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals) or any other known method forgenerating monoclonal antibodies, high affinity chimeric antibodies toPCSK9 are initially isolated having a human variable region and a mouseconstant region. The VELOCIMMUNE® technology involves generation of atransgenic mouse having a genome comprising human heavy and light chainvariable regions operably linked to endogenous mouse constant regionloci such that the mouse produces an antibody comprising a humanvariable region and a mouse constant region in response to antigenicstimulation. The DNA encoding the variable regions of the heavy andlight chains of the antibody are isolated and operably linked to DNAencoding the human heavy and light chain constant regions. The DNA isthen expressed in a cell capable of expressing the fully human antibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. The antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc, using standard procedures known tothose skilled in the art. The mouse constant regions are replaced with adesired human constant region to generate the fully human antibody ofthe invention, for example wild-type or modified IgG1 or IgG4. While theconstant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

In general, the antibodies that can be used in the methods of thepresent invention possess high affinities, as described above, whenmeasured by binding to antigen either immobilized on solid phase or insolution phase. The mouse constant regions are replaced with desiredhuman constant regions to generate the fully human antibodies of theinvention. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Specific examples of human antibodies or antigen-binding fragments ofantibodies that specifically bind PCSK9 which can be used in the contextof the methods of the present invention include any antibody orantigen-binding fragment which comprises the three heavy chain CDRs(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region(HCVR) having an amino acid sequence selected from the group consistingof SEQ ID NOs: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114,118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218,234, 238, 242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334,338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434, 450,454, 458, 474, 478, 482, 498, 502, 506, 522, 526, 530, 546, 550, 554,570, 574, 578, 594, 598, 602, 618, 622, 626, 642, 646, 650, 666, 670,674, 690, 694, 698, 714, 718, 722, 738 and 742, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity. The antibody or antigen-bindingfragment may comprise the three light chain CDRs (LCVR1, LCVR2, LCVR3)contained within a light chain variable region (LCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 10, 20,24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144,154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260,264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370,380, 384, 394, 404, 408, 418, 428, 432, 442, 452, 456, 466, 476, 480,490, 500, 504, 514, 524, 528, 538, 548, 552, 562, 572, 576, 586, 596,600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682, 692, 696, 706,716, 720, 730, 740 and 744, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises the six CDRs (HCDR1, HCDR2,HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy and light chain variableregion amino acid sequence pairs (HCVR/LCVR) selected from the groupconsisting of SEQ ID NOs: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48,50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 981106, 114/116, 118/120,122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188,190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250,258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312,314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380,382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442,450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504,506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572,574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634,642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696,698/706, 714/716, 718/720, 722/730, 738/740 and 742/744.

In certain embodiments of the present invention, the anti-PCSK9antibody, or antigen-binding fragment thereof, that can be used in themethods of the present invention has HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3amino acid sequences selected from SEQ ID NOs: 76/78/80/84/86/88(mAb316P) and 220/222/224/228/230/232 (mAb300N) (See US Patent App. PublNo. 2010/0166768).

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises HCVR/LCVR amino acid sequencepairs selected from the group consisting of SEQ ID NOs: 2/10, 18/20,22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94196,98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164,166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226,234/236, 238/240, 242/250, 258/260, 2621264, 266/274, 282/284, 286/288,290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356,358/360, 362/370, 378/380, 3821384, 386/394, 402/404, 406/408, 410/418,426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480,482/490, 498/500, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548,550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610,618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672,674/682, 690/692, 694/696, 6981706, 7141716, 718/720, 722/730, 738/740and 742/744.

Pharmaceutical Compositions and Methods of Administration

The present invention includes methods which comprise administering aPCSK9 inhibitor to a patient, wherein the PCSK9 inhibitor is containedwithin a pharmaceutical composition. The pharmaceutical compositions ofthe invention are formulated with suitable carriers, excipients, andother agents that provide suitable transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30 ™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and 111 (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by knownmethods. For example, the injectable preparations may be prepared, e.g.,by dissolving, suspending or emulsifying the antibody or its saltdescribed above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

Dosage

The amount of PCSK9 inhibitor (e.g., anti-PCSK9 antibody) administeredto a subject according to the methods of the present invention is,generally, a therapeutically effective amount. As used herein, thephrase “therapeutically effective amount” means a dose of PCSK9inhibitor that results in a detectable reduction in serum Lp(a). Forexample, “therapeutically effective amount” of a PCSK9 inhibitorincludes, e.g., an amount of PCSK9 inhibitor that causes a reduction ofat least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more in Lp(a)levels when administered to a human patient, e.g., as illustrated inExample 2, herein. Alternatively, animal models can be used to establishwhether a particular amount of a candidate PCSK9 inhibitor is atherapeutically effective amount.

In the case of an anti-PCSK9 antibody, a therapeutically effectiveamount can be from about 0.05 mg to about 600 mg, e.g., about 0.05 mg,about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg,about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220mg, about 230 mg, about 240 mg, about 250 wig, about 260 mg, about 270mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570mg, about 580 mg, about 590 mg, or about 600 mg, of the anti-PCSK9antibody.

The amount of anti-PCSK9 antibody contained within the individual dosesmay be expressed in terms of milligrams of antibody per kilogram ofpatient body weight (i.e., mg/kg). For example, the anti-PCSK9 antibodymay be administered to a patient at a dose of about 0.0001 to about 10mg/kg of patient body weight.

Combination Therapies

The methods of the present invention, according to certain embodiments,may comprise administering a pharmaceutical composition comprising ananti-PCSK9 antibody to a patient who is on a therapeutic regimen for thetreatment of hypercholesterolemia at the time of, or just prior to,administration of the pharmaceutical composition of the invention. Forexample, a patient who has previously been diagnosed withhypercholesterolemia may have been prescribed and is taking a stabletherapeutic regimen of another drug prior to and/or concurrent withadministration of a pharmaceutical composition comprising an anti-PCSK9antibody. The prior or concurrent therapeutic regimen may comprise,e.g., (1) an agent which induces a cellular depletion of cholesterolsynthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A(CoA) reductase, such as a statin (e.g., cerivastatin, atorvastatin,simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin,pravastatin, etc.); (2) an agent which inhibits cholesterol uptake andor bile acid re-absorption; (3) an agent which increase lipoproteincatabolism (such as niacin); and/or (4) activators of the LXRtranscription factor that plays a role in cholesterol elimination suchas 22-hydroxycholesterol. In certain embodiments, the patient, prior toor concurrent with administration of an anti-PCSK9 antibody is on afixed combination of therapeutic agents such as ezetimibe plussimvastatin; a statin with a bile resin (e.g., cholestyramine,colestipol, colesevelam); niacin plus a statin (e.g., niacin withlovastatin); or with other lipid lowering agents such as omega-3-fattyacid ethyl esters (for example, omacor).

Administration Regimens

According to certain embodiments of the present invention, multipledoses of a PCSK9 inhibitor may be administered to a subject over adefined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of a PCSK9 inhibitor. As used herein, “sequentially administering”means that each dose of PCSK9 inhibitor is administered to the subjectat a different point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentinvention includes methods which comprise sequentially administering tothe patient a single initial dose of a PCSK9 inhibitor, followed by oneor more secondary doses of the PCSK9 inhibitor, and optionally followedby one or more tertiary doses of the PCSK9 inhibitor.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the PCSK9 inhibitor. Thus,the “initial dose” is the dose which is administered at the beginning ofthe treatment regimen (also referred to as the “baseline dose”); the“secondary doses” are the doses which are administered after the initialdose; and the “tertiary doses” are the doses which are administeredafter the secondary doses. The initial, secondary, and tertiary dosesmay all contain the same amount of PCSK9 inhibitor, but will generallydiffer from one another in terms of frequency of administration. Incertain embodiments, however, the amount of PCSK9 inhibitor contained inthe initial, secondary and/or tertiary doses will vary from one another(e.g., adjusted up or down as appropriate) during the course oftreatment.

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more) days after the immediately preceding dose.The phrase “the immediately preceding dose,” as used herein, means, in asequence of multiple administrations, the dose of PCSK9 inhibitor whichis administered to a patient prior to the administration of the verynext dose in the sequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof a PCSK9 inhibitor. For example, in certain embodiments, only a singlesecondary dose is administered to the patient. In other embodiments, twoor more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses areadministered to the patient. Likewise, in certain embodiments, only asingle tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to29 days after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 1 to 60days after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human PCSK9

Human anti-PCSK9 antibodies were generated as described in US PatentApp. Publ No. 2010/0166768. The exemplary PCSK9 inhibitor used in thefollowing Example is the human anti-PCSK9 antibody designated mAb316P.mAb316P has the following amino acid sequence characteristics: heavychain variable region (HCVR) comprising SEQ ID NO:90; light chainvariable domain (LCVR) comprising SEQ ID NO:92; heavy chaincomplementarity determining region 1 (HCDR1) comprising SEQ ID NO:76;HCDR2 comprising SEQ ID NO:78; HCDR3 comprising SEQ ID NO:80; lightchain complementarity determining region 1 (LCDR1) comprising SEQ IDNO:84; LCDR2 comprising SEQ ID NO:86; and LCDR3 comprising SEQ ID NO:88.

Example 2 Clinical Trials of an Anti-PCSK9 Monoclonal Antibody, as Mono-or Add-on Therapy in Heterozygous Familial and Non-FamilialHypercholesterolemia Introduction

This example describes the results from two clinical trials in whichsingle doses of the anti-PCSK9 monoclonal antibody mAb316P wereadministered intravenously (IV) or subcutaneously (SC) to healthyvolunteers, and a multiple-dose clinical trial of mAb316P in subjectswith either Familial Hypercholesterolemia (FH) or nonFH, receivingstable doses of atorvastatin or dietary intervention only.

Methods

A. Study Design

Three clinical trials were conducted using mAb316P administered to humanpatients. Two of the trials were single dose placebo-controlled studiesof mAb316P administered IV or SC, respectively. The third trial was aphase 1b, double-blind, randomized, placebo-controlled, ascendingmultiple-dose evaluation of mAb316P administered SC to subjects with FHor nonFH. The third trial was conducted in two parts: Part A and Part B.In Part A of the third trial, each subject received a total of 3administrations of mAb316P (50 mg, 100 mg or 150 mg) or matchingplacebo. In Part B of the third trial, each subject received 2administrations of either mAb316P (200 mg) or matching placebo. In bothparts of the multiple SC dose trial, the first dose was administered inan inpatient unit where subjects were confined and observed for 48 hourspost-dose. Subsequent doses were administered in an outpatient settingwith at least 2 hours of post-dose observation.

B. Dose and Dose Escalation

The single dose IV study included 5 sequential cohorts (0.3, 1.0, 3.0,6.0, or 12.0 mg/kg of mAb316P), and the single SC dose trial included 4sequential dose cohorts (50, 100, 150, and 250 mg of mAb316P). Based oninitial results from these trials, the third trial evaluated SC mAb316Pdoses of 50 mg, 100 mg, 150 mg, or placebo administered on days 1, 29and 43 to 7 groups of subjects with FH or nonFH (Part A), and 200 mg orplacebo administered on days 1 and 29 to an eighth group of subjectswith FH or nonFH (Part B). The dosing regimens and patient compositionsof the three trials are summarized in Table 1.

TABLE 1 Total No. mAb 316 Patient Patients Patient ScreeningAtorvastatin Dose Group (mAb316:Pbo) Type LDL-C Dose Single IV Dose 0.3mg/kg 1  8 (6:2) Healthy >100 mg/dL None 1.0 mg/kg 2 Volunteers 3.0mg/kg 3 6.0 mg/kg 4 12.0 mg/kg 5 Single SC Dose 50 mg 1  8 (6:2)Healthy >100 mg/dL None 100 mg 2 Volunteers 150 mg 3 250 mg 4 MultipleSC Dose Part A 50 mg 1  7 (5:2) FH >100 mg/dL 10-40 mg OD 2 10 (8:2)nonFH 100 mg 3  7 (5:2) FH 4 10 (8:2) nonFH 150 mg 5  7 (5:2) FH 6 10(8:2) nonFH 7 10 (8:2) nonFH >130 mg/dL None Part B 200 mg 8 10 (8:2) FH& nonFH >100 mg/dL 10-40 mg OD

In the multiple SC dose trial, each dose level commenced with a sentinelgroup of 3 (FH, non-FH, or both) subjects, with at least 1, but not morethan 2, subjects receiving mAb316P. Enrollment of additional subjects ata given dose level only proceeded after the initial 3 subjects hadsafely completed their day 3 post-treatment assessments. Enrollment inthe next higher dose level was opened once 10 subjects had completed theday 15 safety assessments. Enrollment, once started at each dose level,continued independent of the dose escalation decision and day 15 safetyreview.

C. Subjects

Subjects in the single-dose studies were healthy men and women (18-65years of age, 50-95 kg in body weight, body mass index [BMI]=18-30kg/m²) with serum LDL-C>100 mg/dL (2.59 mmol/L). The use of allnon-study agents to alter lipids was prohibited throughout thesestudies.

Subjects in the multiple-dose study had heterozygous FH or nonFH (18-65years of age, BMI=18.0-35.0 kg/m²) and were on stable atorvastatintherapy (10 to 40 mg per day) with LDL cholesterol>100 mg/dL (2.59mmol/L), or nonFH and were on diet only with LDL cholesterol>130 mg/dL(3.36 mmol/L) (Table 1), All subjects reviewed and signed an informedconsent previously approved by an institutional review board prior toany study related procedures. Subjects in the FH group met Simon Broomecriteria for definite or possible FH (Simon Broome Register Group(1991), BMJ 303:893-896; Neil et al., (2000), BMJ 321:148) while nonFHsubjects did not. Those taking atorvastatin were on a stable dose, 10 to40 mg daily, for at least 28 days prior to baseline, and remained on thesame dose throughout the study. NonFH patients on diet only could nothave received lipid-lowering therapy for at least 28 days prior tobaseline, and remained off such treatment throughout the study.Potentially eligible patients not on atorvastatin could enter a 4 weekrun-in period during which were switched to a dose of atorvastatin (10to 40 mg per day) that was likely to maintain LDL cholesterol near theirpre-study level and >100 mg/dL (2.59 mmol/L). All subjects hadtriglycerides≦300 mg/dL and blood pressure controlled on fewer than 3antihypertensive medications. Patients with a history ofcerebrovascular, cardiovascular, or peripheral vascular atheroscleroticdisease, diabetes, or disorders known to produce secondary elevations ofLDL cholesterol were excluded as were those with hepatic transaminases(ALT or AST)>1.2 times the upper limit of normal (×ULN), >2+proteinuriaor creatine kinase (CK)>3×ULN, unless clearly exercise related in whichcase they were required to have repeat testing with CK<3×ULN.

D. Laboratory Measurements and Methods

In the multi-dose study, serum lipid (total-, HDL-, LDL-, non-HDL-, andVLDL cholesterol) and apolipoprotein (Apo) B, A1 and Lp(a), hs-CRP andsafety laboratory tests were performed after 12 hour overnight fasts(water only) on screening (day −21 to −3), day −2 (for those onatorvastatin), day −1, days 1, 2, 3, 8, 15, 29, 43, 57, 71, 85, 99, 120,and the end-of-study (day 148). All laboratory measurements wereperformed by a central laboratory which maintained Part IIIcertification by CDC Lipid Standardization Program and accreditation bythe College of American Pathologists. Triglycerides and cholesterol weremeasured with enzymatic colorimetric tests (Olympus AU2700 or AU5400Analyzer, Olympus, Center Valley, Pa., USA) with calibration directlytraceable to CDC reference procedures. Apo B containing lipoproteinswere precipitated with dextran sulphate and HDL cholesterol was measuredon the supernatant. (Warnick et al., (1978), J. Lipid Res. 19:65-76).LDL- and VLDL cholesterol were measured after preparativeultracentrifugation (beta-quantification). Apo A1, B, Lp(a) and hs-CRPwere measured with rate immune-nephelometry (Dade Behring BNIInephelometer, Siemens Healthcare Diagnostics, Deerfield, Ill., USA). Alllipid, apolipoprotein, and hs-CRP values were blinded to theinvestigators, study staff, and patients from after day −2 for theatorvastatin treated groups or day −1 for diet only group.

E. Statistical Plan

Effects of mAb316P on lipid parameters in the single-dose studies wereassessed using analysis of covariance (ANCOVA) models. Least squaresmeans of differences between treatment groups and the pooled placebogroup, 95% confidence intervals, and p-values for comparison betweentreatment groups versus placebo by visit were obtained within theframework of ANCOVA. Significance for all tests was set at 0.05.

In the multi-dose study, all subjects who received placebo andatorvastatin were pooled into two placebo groups of either FH or nonFHsubjects. The 6 subjects in both the FH and nonFH (atorvastatin treated)placebo groups, and 5 and 8 subjects in each of the respective FH andnonFH mAb316P-treated dose groups provided at least 80% power to detecta treatment difference of 30% (SD=15%) versus placebo in mean percentagechange from baseline of LDL cholesterol at each study visit whencomparing each dose with placebo with a 2-sided test at the 5%significance level. No adjustments were made for multiple comparisons.Group 7 results were summarized separately by treatment and placebo andthe two groups were compared individually. ANCOVA with treatment arm asthe fixed effect and the relevant baseline value as a covariate was usedto analyze the continuous variables. Missing values were imputed by theLast Observation Carried Forward (LOCF) method. All p-values, except fortriglycerides and Lp(a), which were derived from the Rank-based analysisof covariates, were drawn from the ANCOVA model.

To summarize the lipid and lipoprotein effects, results were selectedfrom study day 57 on which the effects of two subsequent 2-week dosingperiods could be observed. The study was not powered for a directstatistical comparison of the response of FH subjects to nonFH subjects.

Results

A. Study Population

A total of 40 subjects were randomized in the single dose IV study and32 were randomized in the single dose SC study. For Part A of themultiple-dose SC study, a total of 97 subjects were screened and 62 wererandomized (21 FH and 41 nonFH). A total of 10 subjects were included inPart B of the multi-dose SC study (4 FH and 6 nonFH). Baselinecharacteristics of the subjects in the single dose studies and Part A ofthe multi-dose study are shown in Table 2A. Baseline characteristics ofthe subjects in Part B of the multi-dose study are shown in Table 2B.

TABLE 2A Multiple Dose SC - Part A Single Single nonFH Diet Dose DoseAlone (no IV SC FH nonFH atorvastatin) No. Patients 40 32 21 30 10Median Age 36 34 40 52 52 Gender Male 65% 74% 81% 59% 56% Female 35% 26%19% 41% 44% Median BMI 28 25 27 27 27 (kg/m²) Race (%) White 55% 44% 86%93% 100%  Black/African 37.5%   50% 14%  7%  0% American AmericanIndian/ 7.5%   6%  0%  0%  0% Alaskan Native Atorvastatin Dose 10 mgNone None 14% 63% None 20 mg None None 33% 33% None 40 mg None None 52% 4% None Baseline Value LDL-C (mg/dL) 133 127 134 111 174 ApoB (mg/dL)   1.1 1.0 1.1 1.0 1.3 HDL-C (mg/dL)  54 55 44 51 51 TG (mg/dL) 108 103113 136 133

TABLE 2B Multiple Dose SC - Part B (200 mg dose) FH and NonFH CombinedFH NonFH Pbo mAb316P Pbo mAb316P Pbo mAb316P No. Patients 2 8 1 3 1 5Median Age 35 45.5 27 50 43 41 Gender Male 50% 37.5% 100% 66.7%  0%  20%Female 50% 62.5%  0% 33.3% 100%  80% Median BMI 29.39 26.02 28.26 25.4330.52 26.61 (kg/m²) Race (%) White 100%   100% 100%  100% 100% 100%Atorvastatin Dose 10 mg 50%   75%  0% 33.3% 100% 100% 20 mg  0% 12.5% 0% 33.3%  0%  0% 40 mg 50% 12.5% 100% 33.3%  0%  0% Baseline ValueLDL-C 3.05 3.32 3.76 3.78 2.33 3.08 (mmol/L) ApoB (g/L) 0.85 1.06 0.921.05 0.77 1.07 HDL-C 1.03 1.23 0.88 1.19 1.17 1.30 (mmol/L) TG 1.17 1.231.01 0.98 1.33 1.30 (mmol/L)

B. Lipid and Lipoprotein Response

Administration of a single IV or SC dose of mAb316P to healthyvolunteers produced similar mean maximum percent reductions in LDLcholesterol of 55-60%, The degree and duration of LDL cholesterollowering was dose dependent and LDL cholesterol reductions weresustained at least until 29 and 22 days after administration of higherdose levels of IV or SC mAb316P, respectively. Similarly, in themultiple-dose study, the mean percent reduction from baseline in LDLcholesterol was dose dependent and exceeded 50% two weeks after dosingwith 150 mg in the FH and nonFH on atorvastatin and the nonFH diet alonepopulations. All but 1 subject in each population that received 150 mgin the multiple-dose study experienced a reduction of at least 40% frombaseline.

Lipid and apolipoprotein baseline and day 57 levels for all treatmentgroups in Part A of the multiple-dose study are shown in Tables 3A(atorvastatin-treated subjects) and 3B (diet only—no atorvastatintreatment).

TABLE 3A Atorvastatin-Treated Subjects Placebo mAb316P Dose (Pbo) 50 mg100 mg 150 mg Parameter N = 12 N = 13 N = 13 N = 13 LDL Baseline 125(19) 114 (15) 121 (31) 123 (27) Cholesterol day 57 195 (17) 148 (29) 130(16) 127 (26) mean (SD) % change vs Pbo −39.2 −53.7 −61.0 mg/dL p valuevs Pbo <0.0001 <0.0001 <0.0001 Total Baseline 193 (20) 191 (24) 192 (27)197 (32) Cholesterol day 57 195 (17) 148 (29) 130 (16) 127 (26) mean(SD) % change vs Pbo −24.6 −33.2 −36.4 mg/dL p value vs Pbo <0.0001<0.0001 <0.0001 HDL Baseline  43 (10)  52 (16)  48 (10)  48 (11)Cholesterol day 57  42 (10)  55 (14)  51 (10)  54 (12) mean (SD) %change vs Pbo 13.2 11.3 18.2 mg/dL p value vs Pbo 0.0153 0.0336 0.0009LDL Baseline 125 (20) 115 (16) 119 (30) 123 (29) Cholesterol day 57 129(15)  74 (15)  58 (14)  52 (21) mean (SD) % change vs Pbo −41.7 −55.8−62.4 mg/dL p value vs Pbo <0.0001 <0.0001 <0.0001 TriglyceridesBaseline    113 (60:244)    118 (43:171)    135 (46:210)    117 (65:278)median day 57    111 (55:186)    97 (40:189)    111 (31:173)    92(60:181) (min:max) % change vs Pbo −16.3 −7.5 −11.7 mg/dL p value vs Pbo0.0142 0.2515 0.0609 Apolipo- Baseline 107 (12) 104 (15) 100 (21) 106(22) protein B day 57 108 (14)  74 (14)  60 (12)  58 (14) mean (SD) %change vs Pbo −31.5 −42.0 −46.4 mg/dL p value vs Pbo <0.0001 <0.0001<0.0001 Apolipo- Baseline 132 (19) 155 (36) 144 (23) 145 (23) protein A1day 57 132 (20) 162 (31) 150 (20) 161 (24) mean (SD) % change vs Pbo 9.96.9 13.5 mg/dL p value vs Pbo 0.019 0.086 0.013 Lipoprotein (a) Baseline   22 (2:121)   46 (5:151)   50 (7:142)   61 (5:154) median (min:max)day 57    13 (2:119)   42 (4:145)   26 (3:95)   47 (5:119) mg/dL %change vs Pbo −15.5 −24.1 −18.3 p value vs Pbo 0.1109 0.0015 0.1226

TABLE 3B Diet Only - No Atorvastatin Treatment Placebo (Pbo) 150 mgmAb316P Parameter N = 2 N = 8 LDL Baseline 152 (16) 179 (49) Cholesterolday 57 242 (45) 154 (29) mean (SD) % change vs Pbo −57.0 mg/dL p valuevs Pbo <0.0001 0.0023 Total Baseline 228 (6)  257 (58) Cholesterol day57 242 (45) 154 (29) mean (SD) % change vs Pbo −43.3 mg/dL p value vsPbo 0.0025 HDL Baseline  54 (14 50 (8) Cholesterol day 57 64 (8)  51(10) mean (SD) % change vs Pbo −18.9 mg/dL p value vs Pbo 0.17 LDLBaseline 152 (12) 177 (49) Cholesterol day 57 159 (30)  79 (24) mean(SD) % change vs Pbo −58.4 mg/dL p value vs Pbo <0.0025 TriglyceridesBaseline    116 (86:146)    127 (82:268) median day 57    93 (68:118)   116 (70:186) (min:max) % change vs Pbo −16.86 mg/dL p value vs Pbo0.4820 Apolipoprotein Baseline 118 (23) 138 (37) B mean (SD) day 57 118(6)   76 (19) mg/dL % change vs Pbo −45.0 p value vs Pbo 0.003Apolipoprotein Baseline 138 (6)  151 (15) A1 mean (SD) day 57 170 (32)154 (16) mg/dL % change vs Pbo −19.2 p value vs Pbo 0.073 LipoproteinBaseline    47 (30:63)   34 (5:111) (a) median day 57    58 (41:75)   39(4:119) (min:max) % change vs Pbo −43.8 mg/dL p value vs Pbo 0.0415

The LDL cholesterol response at day 57 was similar in all subjectsirrespective of FH or nonFH, atorvastatin treated or on diet alone.Corresponding changes were also observed in total- and non-HDLcholesterol and Apo B (Tables 3A and 3B). Importantly, reductions werealso observed in Lp(a). Additionally, in patients taking atorvastatinfavorable changes were seen in both HDL cholesterol and apoA1.

Similar improvements were observed in the mAb316P-treated patients inPart B of the multiple-dose study, That is, subcutaneous administrationof 200 mg of mAb316P induced rapid, substantial, and sustainedreductions in LDL-C, total cholesterol, non-HDL-C, apoB, and in theratio of apoB/apoA. Patients who received 200 mg of mAb316P SC alsoappeared to demonstrate a trend toward higher levels of HDL-C and ApoA1.

Discussion

This Example describes human trials of the anti-PCSK9 antibody referredto herein as mAb316P, starting with two single ascending dose studies inhealthy volunteers and extending into a large multiple-dose proof ofconcept trial in patients with both FH and nonFH treated with either astatin or on diet alone. These trials confirm the potential to bringabout rapid and significant reductions in LDL cholesterol with PCSK9inhibition in both familial and nonfamilial hypercholesterolemia,whether on a statin or on diet alone. The populations tested encompassthe large majority of patients with hypercholesterolemia.

The robust and reproducible effect on LDL cholesterol of mAb316P fromsingle IV dosing, through single SC dosing, to multiple SC doses is seenwithin an unexpectedly short time after administration, reaching amaximum at approximately 2 weeks. This rapidity exceeds that for allother therapeutic modalities for hypercholesterolemia other than removalof LDL cholesterol via apheresis, and is more rapid than for statins.That large reductions of LDL cholesterol can be achieved with mAb316P inpatients already on relatively high doses of atorvastatin known toproduce 40% to 50% LDL cholesterol reductions clearly distinguishes thepotential of mAb316P from other investigational therapies that have beenadded to statins, such as ezetimibe, bile acid sequestrants, andsqualene synthase inhibitors. The LDL cholesterol lowering efficacy ofmAb316P, combined with apparent tolerability and safety, highlights thesurprising therapeutic advantages of mAb316P over several other ApoB/LDL-C lowering agents in development such as Apo B antisense, MTPinhibitors, and thyroid hormone analogues. In fact, the achievement inmany of the subjects in these studies of low LDL cholesterol levels of50 mg per deciliter (1.29 mmol/L) or less without any increase inhepatic transaminases is in marked contrast to results obtained withagents that inhibit hepatic VLDL and LDL formation. Epidemiology studiesdemonstrating that genetic under-expression or even the absence of PCSK9and life-long low levels of LDL cholesterol appear not to be associatedwith unexpected morbidity or mortality, provide a level of comfort withregard to the therapeutic potential of drug inhibition of PCSK9.

The anti-PCSK9 antibodies of the present invention such as mAb316P alsoprovide patients unable to tolerate statins (now estimated to beapproximately 5% to 10% of all statin treated patients) a significantopportunity to achieve large (at least 50%) reductions in LDLcholesterol. For many of the estimated 10 million patients worldwidewith FH, the anti-PCSK9 antibodies of the invention, including mAb316P,offer the real possibility that their use along with statins may finallyachieve optimal LDL cholesterol control and potentially further reducetheir substantial risk of the early and recurrent cardiovasculardisease. The currently disclosed therapeutic methodologies may alsooffer many patients the potential to discontinue LDL apheresis, aninvasive, time consuming, expensive every 2-week procedure that providesonly short-term LDL cholesterol reduction.

Additional unexpected beneficial effects on other lipids were seen inHDL cholesterol, Apo A1 and, most surprisingly, Lp(a) which trended to,or reached, statistical significance, including in patients treated withatorvastatin. Importantly, before now, no therapeutic biologic agents(e.g., anti-PCSK9 antibodies) have been shown clinically to reduceLp(a), which has been thought not to be cleared via the LDL receptor.Thus, the current experiments are the first demonstration of the abilityof a PCSK9 inhibitor to reduce serum Lp(a) levels in patients.

Regarding safety, mAb316P injection site reactions were minimal, mAb316Pwas also generally safe and well tolerated with no trend in drug-relatedadverse events and no evidence of hepato- or myo-toxicity. The few CKelevations that were seen were exercise related.

In summary, this Example shows that mAb316P inhibition of PCSK9, besideseffectively lowering LDL cholesterol in human patients, surprisinglyreduced Lp(a) levels as well.

Example 3 A Randomized, Double-Blind, Placebo Controlled, 12 Week Studyof the Safety and Efficacy of an Anti-PCSK9 Monoclonal Antibody inPatients with Heterozygous Familial Hypercholesterolemia

A clinical trial was conducted to assess the efficacy of varyingsubcutaneous (SC) doses and dosing regimens of mAb316P on serumlow-density lipoprotein cholesterol (LDL-C) and otherlipids/apolipoproteins (e.g., total cholesterol, high-densitylipoprotein cholesterol, triglycerides, apo B, Apo A1, and Lp[a]) inpatients with heterozygous familial hypercholesterolemia (heFH).

Patient Population

The patient population for this study included men and women, ages 18-75years old, who were diagnosed with heFH, exhibited LDL-C levels of 100mg/dL or greater, and who were on a stable daily statin dose (eitherwith or without ezetimibe) at the start of the study. Patients wereexcluded if they were taking any additional lipid lowering compoundssuch as fibrates, niacin, omega-3 fatty acids, bile acid resins, plantstanols (e.g., Benecol, flaxseed oil, psyllium), or red yeast rice. Atotal of 77 patients completed the study.

Drug Formulation

The drug formulation comprised 150 mg/mL of mAb316P, 10 mM histidine,0.2% polysorbate 20, 10% sucrose, and had a pH of 6.0.

Method of Administration

The drug formulation (or placebo) was administered to the patients bysubcutaneous injection into the abdomen. Each treatment included twosubcutaneous injections of 1 mL each.

Administration Regimens

The patients were divided into five groups. Group 1 included 15 patientswho received placebo once every two weeks; Group 2 included 16 patientswho received 150 mg of mAb316P once every two weeks; Group 3 included 15patients who received 150 mg of mAb316P once every four weeks; Group 4included 16 patients who received 200 mg of mAb316P once every fourweeks; and Group 5 included 15 patients who received 300 mg of mAb316Ponce every four weeks. The study duration was 12 weeks.

Efficacy Assessment

Blood samples were collected from the patients throughout the study, andduring an 8 week follow-up period. The samples were collected after atleast a 12 hour fast (in the morning before any drug intake).Measurements of the following parameters were determined from the bloodsamples: (1) Total Cholesterol; (2) LDL-C; (3) HDL-C; (4) Triglycerides(TG); (5) Apo B; (6) Apo A1; and (7) Lp(a). The median percent changesin these parameters compared to placebo at the end of the study (week12) are summarized in Table 4.

TABLE 4 Group 1 2 3 4 5 No. of Patients 15 16 15 16 15 Treatment PlacebomAb316P mAb316P mAb316P mAb316P Dose N/A 150 mg 150 mg 200 mg 300 mgDosing Q2W Q2W Q4W Q4W Q4W Frequency Total −6.52 −38.27 −18.89 −15.31−28.84 Cholesterol LDL-C −4.90 −66.71 −24.30 −24.07 −49.35 HDL-C +2.48+14.63 +6.33 +6.53 +4.48 Triglycerides −10.55 −16.23 −16.73 −9.87 −4.92Apo B −7.32 −48.97 −19.09 −14.69 −36.27 Apo A1 −7.24 +9.98 +3.13 +0.33+5.39 Lp(a) −11.07 −22.50 −11.30 −8.00 −14.29

Example 4 A Randomized, Double-Blind, Parallel-Group, PlaceboControlled, 12 Week Study of the Safety and Efficacy of an Anti-PCSK9Monoclonal Antibody in Patients with Primary Hypercholesterolemia onStable Atorvastatin Therapy

A clinical trial was conducted to assess the efficacy of five doses andtwo dose regimens of mAb316P over 12 weeks in patients with primaryhypercholesterolemia and LDL-C levels greater than 100 mg/dL on ongoingstable atorvastatin therapy. Efficacy was assessed based on changes inserum low-density lipoprotein cholesterol (LDL-C) and otherlipids/apolipoproteins (e.g., total cholesterol, high-densitylipoprotein cholesterol, triglycerides, apo B, Apo A1, and Lp[a]) overthe course of the study.

Patient Population

The patient population for this study included men and women, ages 18-75years old, who were diagnosed with primary hypercholesterolemia,exhibited LDL-C levels of 100 mg/dL or greater, and who were on stableatorvastatin therapy of 10, 20 or 40 mg for at least six weeks prior tothe start of the study. Patients were excluded if they were taking anyadditional lipid lowering compounds such as fibrates, niacin, omega-3fatty acids, bile acid resins, plant stanols (e.g., Benecol, flaxseedoil, psyllium), or red yeast rice. A total of 118 patients completed thestudy.

Drug Formulation

The drug formulation comprised 150 mg/mL of mAb316P, 10 mM histidine,0.2% polysorbate 20, 10% sucrose, and had a pH of 6.0.

Method of Administration

The drug formulation (or placebo) was administered to the patients bysubcutaneous injection into the abdomen. Each treatment included twosubcutaneous injections of 1 mL each.

Administration Regimens

The patients were divided into six groups. Group 1 included 20 patientswho received placebo once every two weeks; Group 2 included 19 patientswho received 50 mg of mAb316P once every two weeks; Group 3 included 20patients who received 100 mg of mAb316P once every two weeks; Group 4included 18 patients who received 150 mg of mAb316P once every twoweeks; Group 5 included 20 patients who received 200 mg of mAb31P onceevery four weeks; and Group 6 included 21 patients who received 300 mgof mAb316P once every four weeks. The study duration was 12 weeks.

Efficacy Assessment

Blood samples were collected from the patients throughout the study, andduring an 8 week follow-up period. The samples were collected after atleast a 12 hour fast (in the morning before any drug intake).Measurements of the following parameters were determined from the bloodsamples: (1) Total Cholesterol; (2) LDL-C; (3) HDL-C; (4) Triglycerides(TG); (5) Apo B; (6) Apo A1; and (7) Lp(a). The median percent changesin these parameters compared to placebo at the end of the study (week12) are summarized in Table 5.

TABLE 5 Group 1 2 3 4 5 6 No. of Patients 20 19 20 18 20 21 TreatmentPlacebo mAb316P mAb316P mAb316P mAb316P mAb316P Dose N/A 50 mg 100 mg150 mg 200 mg 300 mg Dosing Frequency Q2W Q2W Q2W Q2W Q4W Q4W TotalCholesterol −3.73 −23.34 −40.21 −45.03 −29.51 −33.48 LDL-C −6.92 −37.04−64.28 −74.83 −49.46 −51.98 HDL-C +1.81 +5.08 +7.93 +8.96 +5.59 +12.09Triglycerides +22.42 −7.85 −11.41 −22.93 +1.27 −9.17 Apo B +0.76 −28.67−48.67 −54.16 −30.67 −32.95 Apo A1 +0.58 +1.40 −0.88 +1.11 +0.66 +3.42Lp(a) 0.00 −13.33 −27.27 −28.57 −18.92 −11.11

Example 5 A Randomized, Double-Blind, Parallel-Group, PlaceboControlled, Fixed Dose Study of the Safety and Efficacy of an Anti-PCSK9Monoclonal Antibody Co-Administered with 80 mg Atorvastatin in Patientswith Primary Hypercholesterolemia

A clinical trial was conducted to assess the efficacy of mAb316P whenco-administered with 80 mg of atorvastatin over 8 weeks in patients withprimary hypercholesterolemia and LDL-C levels greater than 100 mg/dL.Efficacy was assessed based on changes in serum low-density lipoproteincholesterol (LDL-C) and other lipids/apolipoproteins (e.g., totalcholesterol, high-density lipoprotein cholesterol, triglycerides, apo B,Apo A1, and Lp[a]) over the course of the study.

Patient Population

The patient population for this study included men and women, ages 18-75years old, who were diagnosed with primary hypercholesterolemia,exhibited LDL-C levels of 100 mg/dL or greater, and who were on stableatorvastatin therapy of 10 mg for at least six weeks prior to the startof the study. Patients were excluded if they were taking any additionallipid lowering compounds such as fibrates, niacin, omega-3 fatty acids,bile acid resins, plant stanols (e.g., Benecol, flaxseed oil, psyllium),or red yeast rice. A total of 88 patients completed the study.

Drug Formulation

The drug formulation comprised 150 mg/mL of mAb316P, 10 mM histidine,0.2% polysorbate 20, 10% sucrose, and had a pH of 6.0.

Method of Administration

The drug formulation (or placebo) was administered to the patients bysubcutaneous injection into the abdomen. Each treatment included onesubcutaneous injection of 1 mL.

Administration Regimens

The patients were divided into three groups. Group 1 included 29patients who received placebo once every two weeks plus 80 mgatorvastatin daily; Group 2 included 30 patients who received 150 mg ofmAb316P once every two weeks plus 80 mg atorvastatin daily; and Group 3included 29 patients who received 150 mg of mAb316P once every two weeksplus 10 mg atorvastatin daily. The study duration was 8 weeks.

Efficacy Assessment

Blood samples were collected from the patients throughout the study, andduring an 8 week follow-up period. The samples were collected after atleast a 12 hour fast (in the morning before any drug intake).Measurements of the following parameters were determined from the bloodsamples: (1) Total Cholesterol; (2) LDL-C; (3) HDL-C; (4) Triglycerides(TG); (5) Apo B; (6) Apo A1; (7) Apo B/Apo A1 ratio; and (8) Lp(a). Thepercent changes in these parameters compared to placebo at the end ofthe study are summarized in Table 6

TABLE 6 Group 1 2 3 No. of Patients 29 30 29 Treatment Placebo mAb316PmAb316P Dose N/A 150 mg 150 mg Dosing Frequency Q2W Q2W Q2W DailyAtorvastatin 80 mg 80 mg 10 mg Total Cholesterol −16.59 −47.21 −40.45LDL-C −26.87 −70.62 −70.37 HDL-C −5.71 +5.17 +1.39 Triglycerides −11.89−24.67 −3.98 Apo B −12.00 −58.00 −54.39 Apo A1 −5.56 −4.55 −0.69 Lp(a)−2.70 −31.01 −34.65

Taken together, the results from these studies (Examples 3-5) confirmthe ability of mAB316P to effectively lower LDL cholesterol andbeneficially affect other lipid/apolipoprotein parameters in patientswhen administered at various doses and dosing regimens. These clinicalExamples also confirm the unexpected finding that mAB316P is able tosignificantly reduce Lp(a) levels in patients under variouscircumstances.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method for reducing lipoprotein(a) (“Lp(a)”)levels, the method comprising selecting a patient who exhibits elevatedserum Lp(a), and administering to the patient a pharmaceuticalcomposition comprising a PCSK9 inhibitor.
 2. The method of claim 1,wherein the patient is selected on the basis of a serum Lp(a) levelgreater than about 20 mg/dL.
 3. The method of claim 2, wherein thepatient is selected on the basis of a serum Lp(a) level greater thanabout 100 mg/dL.
 4. The method of claim 1, wherein prior to or at thetime of administration of the pharmaceutical composition, the patient isdiagnosed with or identified as being at risk of developing acardiovascular disease or disorder.
 5. The method of claim 4, whereinthe cardiovascular disease or disorder is selected from the groupconsisting of coronary artery disease, acute myocardial infarction,asymptomatic carotid atherosclerosis, stroke, and peripheral arteryocclusive disease.
 6. The method of claim 4, wherein the cardiovasculardisease or disorder is hypercholesterolemia.
 7. The method of claim 6,wherein the hypercholesterolemia is heterozygous FamilialHypercholesterolemia (heFH).
 8. The method of claim 6, wherein thehypercholesterolemia is not Familial Hypercholesterolemia (nonFH). 9.The method of claim 1, wherein prior to or at the time of administrationof the pharmaceutical composition, the patient is diagnosed with oridentified as being at risk of developing a thrombotic occlusive diseaseor disorder.
 10. The method of claim 9, wherein the thrombotic occlusivedisease or disorder is selected from the group consisting of pulmonaryembolism and central retinal vein occlusion.
 11. The method of claim 1,wherein the PCSK9 inhibitor is an antibody or antigen-binding fragmentthereof that specifically binds PCSK9.
 12. The method of claim 11,wherein the pharmaceutical composition comprises 20 mg to 200 mg of thePCSK9 inhibitor.
 13. The method of claim 12, wherein: the pharmaceuticalcomposition comprises 50 mg to 150 mg of the PCSK9 inhibitor.
 14. Themethod of claim 13, wherein the pharmaceutical composition comprises 50mg of the PCSK9 inhibitor.
 15. The method of claim 13, wherein thepharmaceutical composition comprises 100 mg of the PCSK9 inhibitor. 16.The method of claim 13, wherein the pharmaceutical composition comprises150 mg of the PCSK9 inhibitor.
 17. The method of claim 11, wherein theantibody or antigen binding fragment thereof comprises the heavy andlight chain CDRs of a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of SEQ ID NOs: 90/92 and 218/226.
 18. The method ofclaim 17, wherein the antibody or antigen-binding fragment thereofcomprises heavy and light chain CDR amino acid sequences having SEQ IDNOs:220, 222, 224, 228, 230 and
 232. 19. The method of claim 18, whereinthe antibody or antigen-binding fragment thereof comprises an HCVRhaving the amino acid sequence of SEQ ID NO:218 and an LCVR having theamino acid sequence of SEQ ID NO:226.
 20. The method of claim 17,wherein the antibody or antigen-binding fragment thereof comprises heavyand light chain CDR amino acid sequences having SEQ ID NOs:76, 78, 80,84, 86 and
 88. 21. The method of claim 20, wherein the antibody orantigen-binding fragment thereof comprises an HCVR having the amino acidsequence of SEQ ID NO:90 and an LCVR having the amino acid sequence ofSEQ ID NO:92.
 22. The method of claim 11, wherein the antibody orantigen-binding fragment thereof binds to the same epitope on PCSK9 asan antibody comprising heavy and light chain CDR amino acid sequenceshaving SEQ ID NOs:220, 222, 224, 228, 230 and 232; or SEQ ID NOs: 76,78, 80, 84, 86 and
 88. 23. The method of claim 11, wherein the antibodyor antigen-binding fragment thereof competes for binding to PCSK9 withan antibody comprising heavy and light chain CDR amino acid sequenceshaving SEQ ID NOs:220, 222, 224, 228, 230 and 232; or SEQ ID NOs: 76,78, 80, 84, 86 and
 88. 24. The method of claim 1, wherein the patient ison a therapeutic statin regimen at the time of or just prior toadministration of the pharmaceutical composition.
 25. The method ofclaim 24, wherein the therapeutic statin regimen comprises a statinselected from the group consisting of cerivastatin, atorvastatin,simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin andpravastatin.
 26. The method of claim 25, wherein the statin isatorvastatin.
 27. The method of claim 1, wherein the patient is not on atherapeutic statin regimen at the time of administration of thepharmaceutical composition.